Two series of 22 and 15 atom cyclic enkephalins incorporating a diversely substituted guanidine bridge have been prepared to assess the potential effect of the bridge substitutions on their opioid activity profile. The most notable results were obtained with the shortest cyclic analogues, which showed a significant variation of their binding affinity toward μ and δ opioid receptors in relation to bridge substitution. NMR studies were performed to rationalize these data. Some small analogues were found to exist as at least one major and one minor stable forms, which could be separated by chromatography. In particular, the compounds 13 and 14 with a cyclic substituent were separated in three isomers and the basis of this multiplicity was explored by 2D NMR spectroscopy. All compounds were agonists with slight selectivity for the μ opioid receptor. Compounds 7a (thiourea bridge) and 10a (N-Me-guanidine bridge) showed nanomolar affinity toward μ receptor, the latter being the more selective for this receptor (40-fold).